The human trifunctional enzyme of de novo purine biosynthesis: heterologous expression, purification, and preliminary characterization.
The cDNA for the human trifunctional enzyme of de novo purine biosynthesis, which encodes glycinamide ribonucleotide synthetase, aminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide trans-formylase, has been overexpressed in Escherichia coli and its protein product has been purified to homogeneity. The glycinamide ribonucleotide transformylase activity, which constitutes the C-terminal domain of the trifunctional enzyme, has been characterized with respect to its kinetic constants, Vmax = 3.03 +/- 0.15 micromol/min-mg and Km values for beta-glycinamide ribonucleotide and 10-formyl-5,8-dideazafolate of 0.94 +/- 0.21 and 1.58 +/- 0.25 microM, respectively, and its kinetic mechanism, which is ordered-sequential with the folate substrate binding first. The correspondence of these data to those obtained for the glycinamide ribonucleotide transformylase activity of the mammalian trifunctional enzyme indicates that the recombinant enzyme is fully functional.
Poch
M T
MT
College of Pharmacy, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0004, USA.
Qin
W
W
Caperelli
C A
CA
D019731:Carbon-Nitrogen Ligases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D051026:Phosphoribosylglycinamide Formyltransferase; D011687:Purines / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011687:Purines; D011994:Recombinant Proteins; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D019731:Carbon-Nitrogen Ligases; C022380:phosphoribosylaminoimidazole synthase; C022387:phosphoribosylamine-glycine ligase